Prostate cancer markers

#This application is a National Stage of PCT/EP2012/057225, filed Apr. 19, 2012 which claims priority to European Application No. 11162979.6, filed Apr. 19, 2011, the disclosures of which are incorporated herein by reference in their entirety.

#The present invention is in the field of biology and chemistry. In particular, the invention is in the field of molecular biology. More particular, the invention relates to the analysis of the methylation status of genomic regions. Most particularly, the invention is in the field of diagnosing prostate cancer.

#The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 16, 2013, is named 0117_0002US1_Sequence_Listing.txt and is 234682 bytes in size.

#FIG. 1: Overview of the MeDIP-Seq technology and validation experiments. (A) Schematics of the MeDIP-Seq protocol. Genomic DNA is sheared to a size of 100-200 bp, end repaired and sequencing adapters carrying a distinct barcode are ligated. After denaturation the libraries are incubated with antibodies targeting 5-methylcytosine coupled to magnetic beads. The enriched libraries are amplified and analysed on a SOLiD3+ NGS system using 35 bp fragment sequencing chemistry. (B) Coverage plots of uniquely mapped reads for eight samples (four normal and four tumour) for the CAV2 promoter region (left) and the GSTP1 promoter region (right). Top lane: location of the CpG-island, second lane with CAV2 and GSTP1 labels: coding regions, dark grey: normal, light grey: tumour samples. (C) Bisulphite conversion and mass spectrometry analyses were used for the validation of the MeDIP-Seq results. Left: hypomethylated, promoter associated region in the KLK4-gene (chr19:51411401-51412100), right: hypermethylated intronic region in the PTPRN2-gene (chr7:157360901-157361600). Top 3 samples are derived from tumour DNA, sample 4 to 6 from normal tissues. Line 7: Completely methylated control DNA, line 8: water control. Dark grey circles: no methylation, light grey circles: 100% methylation, white circles mark CpG dinucleotides not accessible for analysis or failed in analysis.

#FIG. 2: Separation of normal (dark grey) and tumour (light grey) samples based on DMRs. (A) Principal component analyses were performed by using the rpm-values of either all bins (upper left corner) or restrictions to either chromosome 8 (bottom row) or the promoter regions (right column) were applied. (B) PAM analyses enabled a selection of seven marker regions used for unsupervised cluster analyses.

#FIG. 3: Differential methylations are due to alterations in tumour cells. Bisulphite analyses of microdissected tumour material. Normal and tumour tissue materials were prepared with macrodissections (light grey bars) or laser captured microdissections (dark grey bars) and bisulphite-MS analyses were performed for 35 differentially methylated regions. Correlation analyses between microdissected and macrodissected samples (A) and unsupervised cluster analyses (B) are shown.

#Aberrant cytosine methylation is among the earliest and most frequent events in the development of prostate cancer. Herein, an immunocapturing approach followed by next generation sequencing was used to generate genome-wide methylation profiles of 51 prostate tumour and 53 normal prostate tissues. Machine-learning techniques identified several sets of differentially methylated genomic regions which enabled a correct classification (100%) of tumour and normal samples.

#It should be noted, that gene names are merely used to constrict the localization of the identified genomic regions. This means that the genomic region must not necessarily form a part of the gene, but lies in the vicinity of that gene. Genomic locations are given in Hg19 annotation.

#Experimental Procedure